1. Field of the Invention
The present invention relates to a process for producing a tissue plasminogen activator.
2. Description of the Prior Art
A tissue plasminogen activator (referred to as tPA hereinafter) has a strong affintiy for the thrombus and hence it readily brings about thrombolysis. Therefore, tPA can be used for the treatment of thrombosis.
At present, urokinase isolated from urine and streptokinase produced by certain strepococci are used as a thrombolytic agent base on plasminogen activation. However, they are poor in affinity for thrombi, and it is necessary to administer in a large dose to obtain the desirable effect. Moreover, they act not only on thrombi but also on fibrinogen and plasminogen in the blood. This leads to side effects such as internal hemorrhage. Under the circumstances, tPA is expected to be a new thrombolytic agent because of its strong affinity for the thrombus, and of thrombolysis in small doses with a minimum of side effect.
The tPA of human origin exists in an extremely small quantity in human normal tissues such as blood vessels, kidneys, and uteri. On the other hand, it can be isolated from the culture medium of a line of human melanoma cells and also from the culture medium of Escherichia coli, yeast, or mammalian cells into which the vector having the gene of human tPA has been introudced by the recombinant DNA technology. It is known that the tPA isolated from the culture medium can be divided into two classes according to the molecular structure. (Rijken, D. C. et al., J. Biol. Chem., Vol. 256 pp 7035-7041, 1981) The original tPA secreted by human melanoma cells has a single chain form composed of 527 amino acids and some sugar chains. In the culture medium, it is converted into tPA of 2-chain form by the action of a protease which exists in the culture medium. This protease cleaves the peptide bond between the 275th arginine and the 276th isoleucine counted from the amino terminus of the peptide chain, to give 2-chain form tPA, with the two strands being joined by one disulfide linkage. Therefore, the tPA isolated in the usual method from the culture medium is a mixture of single chain form tPA and 2-chain form tPA. It is recognized that these two kinds of tPA have a molecular weight of about 69,000 when measured by SDS-polyacrylamide gel electrophoresis under the non-reducing condition. However, under the reducing condition, it is recognized that single-chain form tPA has a molecular weight of about 69,000, and 2-chain form tPA has two values of molecular weight, 36,000 and 33,000.
It is an object of the present invention to provide a process for producing single-chain form tPA which is identical with the naturally occurring tPA. The thus produced tPA will be used as a medicine for medical treatment. This object, however, is not achieved by the conventional cultivation method, because the single-chain form tPA formed in the culture medium is readily converted into 2-chain form tPA by the action of proteases present in the culture medium. Therefore, it is necessary to add an inhibitor for these proteases into the culture medium prior to cultivation. The inhibitor should meet the following requirements. (a) It is effective in inhibiting proteases. (b) It can be easily removed from tPA when needed. (c) It is stable during purification of tPA. (d) It does not interfere with the process analysis and product analysis. (e) It is inexpensive enough to be used in an industrial scale production.
For the production of single-chain form tPA, Collen et al proposd that aprotinin ("Trasylol", a trade name of Bayer Co.), which is a protease inhibitor, should be added to the culture medium and to the buffer solution in the purification process. (Japanese Patent Kokai (Laid-open) No. 28009/82)
Aprotinin is a peptide having a molecular weight of 6500, extracted from bovine lungs and it is a foreign protein to human. Patients injected with aprotinin often suffer from shocks. (See "Yakumu Koho", No. 1271, P. 17, Aug. 11, 1984). Therefore, in case where tPA is used as an injection, it is necessary to remove aprotinin from tPA. completely. However, it is very difficult to remove aprotinin by the usual method without a loss of tPA. In addition, aprotinin is very expensive and it is uneconomical to use aprotinin for the commercial production of tPA. Another disadvantage of using aprotinin is that it interferes with the measurement of tPA activity by Fibrin Clot Lysis Time method (Rijken, D. C. et al., Biochim. Biophys. Acta, Vol. 193, pp 140-153, 1979) and in Fibrin Plate method (Jespersen, J. et al., Haemostasis, Vol. 13, pp 301-315, 1983). Moreover, aprotinin interferes with the determination of protein by Lowry method (Lowry, O. H. et al., J. Biol. Chem., Vol. 193, pp 265-275, 1951) by spectrophotometry at 280 nm.
the present inventors found that single-chain form tPA can be obtained by adding a specific antiplasmin agent, which is a protease inhibitor, to the culture media and all the buffer solutions in the purification process in order to prevent and/or inhibit the conversion of single-chain form tPA to 2-chain form tPA by proteases existing in the culture medium during cultivation process of human melanoma cells or Escherichia coli, yeast or mammalian cells which have been transformed by a vector carrying a gene encoding human tPA and during purification process. The present invention was completed on the basis of these findings.